Project Summary hERG (human eag-related gene; KCNH2) encodes a voltage activated potassium channel expressed in the brain and heart. hERG is the delayed-rectifier (IKr) current that drives repolarization in the cells with long action potentials. Inherited mutations in hERG cause cardiac arrhythmias which can lead to sudden death and dysregulation of hERG is also associated with schizophrenia. Off target effects of pharmaceuticals inhibit hERG and this is the primary mechanism for acquired LQT syndrome, a common clinical problem. Despite the importance of hERG in a myriad of physiological function, how its voltage sensing ability is structurally and functionally coupled to the mechanisms of ion gating is not well understood. This is predominately due to limitations in current methods to study the movements of the S4 helix in response to voltage; the key domain in hERG that senses voltage and transduces the voltage signal to the rest of the channel. To overcome these limitations new techniques are required to uncover the mechanism of S4 and hERG gating. We hypothesize that the S4 helix of hERG undergoes large dynamic movements and indirectly regulates channel closing. To test our hypothesis, we have directly incorporated the small, structurally and functionally non-perturbing, fluorescent non-canonical amino acid L-ANAP in the S4 helix of hERG with amber codon suppression. We implement L-ANAP as powerful fluorescent reporter to rigorously probe short-range conformational changes in hERG channels with transition metal Frster resonance energy transfer (tmFRET). We will measure the dynamic movements of the S4 helix in hERG channels using simultaneous voltage and spectroscopic recordings (tmFRET) to measure the distance of S4 helix movement in response to voltage changes. Second, we will investigate the role of the S4 helix in hERG deactivation(closing) gating by measuring the movement of the S4 helix with tmFRET in fast deactivating hERG channels compared to wildtype channels, and in chemically treated slow deactivating channels. Due to the importance of hERG in cardiac excitability and arrhythmia, determining hERG gating mechanisms directly impacts health and disease.